This grant application is focused on the molecular functions of the proteins encoded by the myc-protooncogene family. Myc proteins are widely expressed during vertebrate development where they regulate cell growth, proliferation, death, and differentiation in response to diverse extracellular signals. Importantly, deregulation of Myc expression is strongly linked to the etiology of many different types of cancers. Myc proteins are sequence-specific DNA binding proteins known to function as part of a transcriptional regulatory network that activates and represses expression of hundreds of target genes. This is accomplished in part by Myc's ability to recruit higher order complexes, several of which act to modify histones and alter chromatin structure. Myc may also possess functions separable from its transcriptional activities. A major goal of this grant is to elucidate in detail the connection between Myc's molecular functions and its biological effects on cell behavior. Aims 1 and 2 explore the mechanisms underlying Myc's roles in pluripotency and differentiation and are based on our preliminary analyses of genomic binding by Myc in murine embryonic stem (ES) cells. In Aim 1 we extend our study of microRNAs induced by c-Myc in ES cells and test the hypothesis that these miRNAs comprise a Myc-regulated pathway that suppresses differentiation. We will determine whether the miRNA-encoding genes are directly regulated by Myc, and employ genetic analysis to define the events in differentiation affected by the miRNAs and their gene targets. We will also identify additional Myc-regulated miRNAs in stem cells. Aim 2 expands on other functional classes of Myc-regulated genes in ES cells. These include genes encoding chromatin modifying factors and as well as genes that are associated with Polycomb repression complexes (PcG). We propose to determine whether Myc regulates PcG function in ES cells and further examine the effects of (i) modulating Myc levels, and (ii) induction of differentiation, on PcG binding, histone modification, and gene expression for a subset of Myc target and other ES cell genes. Aim 3 is focused on a novel form of the Myc protein (Myc-nick) that we identified as a cytoplasmic N-terminal cleavage product of Myc generated by calcium-dependent calpain proteases. Myc-nick lacks a Max dimerization domain and nuclear localization sequences. Our preliminary data suggest that Myc-nick binds to and mediates acetylation of tubulin and promotes differentiation. We propose to characterize the binding of Myc-nick to tubulin, determine the nature of its recruited complexes, and use molecular and genetic approaches to define its role in terminal differentiation. Because many tumors are altered in the capacity to differentiate, delineating the functions of Myc in the context of differentiation is likely to be important in understanding Myc's role in oncogenesis.